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Abstract

Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze the molecular interaction, enabling rapid and repetitive estimation with relatively small amount of sample. Here we describe a general protocol about SPR analysis on the interaction between viral antigen and human MAb (HuMAb) IgG. Anti-human Fcγ is first covalently crosslinked on the sensor chip by amine coupling, and then HuMAb of interest is immobilized via anti-Fcγ MAb IgG interaction as ligand. Antigen injected on the sensor chip causes the SPR change in time course as the result of association and dissociation. By analyzing the kinetics, association rate, dissociation rate, and dissociation constant are obtained.

Imbalance in the refractive index between antigen solution and running buffer will cause high background signal (called bulk effect) and perturb the SPR analysis.

The concentration of antigen is recommended to be 5 μM or higher, because the dissociation constant for weak antigen-antibody interaction sometimes reaches to 10-6 M range and in that case SPR experiment requires relatively high concentration of antigen, that is 5 μM or more.

Inject ligand solutions and determine the optimum pH condition in which the highest response level is observed (Figure 1a).Note: When there is little or no difference, select higher pH in order to avoid ligand deterioration and to promote coupling reaction. Practically, pH 4.0 or 4.5 is selected for immobilization of anti-human Fcγ, though you should determine for your SPR system.

Initialize the chip surface by injecting 30 μl of 50 mM NaOH.

Activate the chip surface by injecting 70 μl of 1:1 EDC/NHS mix.

Inject anti-human Fcγ solution and check the baseline increment. Repeat injection until the increment becomes undetectable.Note: In this step anti-human Fcγ should immobilize as much as possible. Usually, approx. 8,000 (Resonance Unit, RU) increment is obtained.

Inject 70 μl of 1 M 2-ethanolamine-HCl solution to block the active carboxyl groups remained on the sensor chip.Note: If you want stop the experiment in this step, eject the sensor chip from the instrument and store at 4 °C in HBS-EP buffer to prevent drying out.

Check the antigen-MAb interaction and design experiment.

Prepare 1 ml of 1 μg/ml MAb solution by diluting with HBS-EP buffer.Note: The original MAb solution should not include any reagents which perturb the interaction between MAb and anti-human Fcγ, for example, 100 mM glycine buffer of pH 2.4 or lower.

Inject target MAb and check the immobilization amount as the increment of baseline level.Note: For SPR analysis, smaller immobilization amount is preferred as far as the response against antigen gives sufficient signal.

Inject 30 μl of 10 mM glycine solutions of different pH values 1.8, 2.0, 2.2, and 2.4. By checking the decrement of the baseline level, determine the MAb-stripping condition (pH, injection period, number of injections, period required for baseline stabilization).Note: The baseline level should be close to the level before MAb immobilization. An example of practical condition is as follows: Inject 30 μl of 10 mM Glycine pH 2.0 thrice and wash by running buffer for 600 seconds as baseline stabilization period.

Prepare 1 ml of 1 μM antigen solution by diluting with HBS-EP buffer.

Clarify the antigen solution by centrifugation at 15,000 x g for 5 min at room temperature.

Inject 60 μl of the antigen solution and check the SPR response.Note: The minimum response level is 1 RU or less as far as the response curve is recognizable, though it apparently depends on the baseline stability, in other words, stability of the SPR system.

Determine antigen-stripping condition by injecting 60 μl of 1 M NaCl or 3 M MgCl2.Note: As other option, you can remove the whole antigen-MAb with glycine buffer and retry from the MAb immobilization step. Antigen-stripping condition is not necessarily required depending on the experimental design.

Prepare 1 ml of antigen solutions of at least 5 different concentrations by 1:1 serial dilution with HBS-EP buffer.

Determine the experimental design considering the following points.

MAb immobilization condition.

Injection period for the antigen solution and washing period.Notes:

They are corresponding to the lengths of association and dissociation phases, respectively. Set appropriate values to obtain significant signal level and sufficient curvature.

In some cases antigen dissociates very slowly from MAb, and then we need to set very long washing period. When no dissociation is observed, try reducing the immobilizing level of MAb.

Setting for individual flow paths.Note: Usually, SPR system has multiple flow paths useful for blank experiment and for examination of multiple MAbs at once.

Curve analysis

Collected experimental data is analyzed according to the fitting algorism supplied by vendor of the SPR system (For example, see Figure 1c). Usually, 1:1 Langmuir fitting model is employed.Note: Check the chi-square value and quality index supplied by the software.

Figure 1. Practical example of the SPR experiment.

Determination of the pH value for amine-coupling immobilization of anti-human Fcγ. pH 4.0 showed the highest response, however, it also showed signs of leveling off. Then, we have chosen pH 4.5 in this case. Open arrowhead and closed arrowhead indicate the start and end points of the injection of anti-human Fcγ solution, respectively.

Immobilization of MAb on the sensor chip via the interaction between anti-human Fcγ and human MAb. Open arrowhead and closed arrowhead indicate the start and end points of the injection of human MAb solution, respectively. The increment of baseline level (in this case, approx. 10 RU) represents the immobilized amount of MAb.

Representative SPR response for the interaction between influenza B hemagglutinin (HA) and a human anti-HA antibody. Open arrowheads indicate the injection of HA solution and correspond to the association phase. Closed arrowhead indicates the end of the final injection and corresponds to the start of the dissociation phase. Fitting curve obtained by the 1:1 Langmuir fitting model was also shown by gray line.

Notes

Usually SPR analysis is performed under an integrated system in which equipment (sensor chip, buffer bottle, sample tube, etc), experimental design, analysis algorism and even in reagents are all supplied for using that instrument. We just described here a general procedure of SPR experiment for antigen-MAb interaction analysis. Please follow the manual of your instrument and/or contact the vendor for more detailed protocol.

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